lyme borreliosis in europe and north america (epidemiology and clinical practice) || serodiagnosis...

10

SERODIAGNOSIS OFLYME BORRELIOSIS

Barbara J. B. Johnson, Maria E. Aguero-Rosenfeld,and Bettina Wilske

Serology is currently the mainstay of laboratory testing to support the clinical diagnosis

of Lyme borreliosis. Serologic assays provide indirect evidence of Borrelia burgdorferi

infection by detecting the humoral immune response, usually immunoglobulins M and

G, to antigens of the organism. Assays of the immune responses to an infectious agent

do not detect the agent itself, of course, and may be negative when an infected

individual has not yet had time to develop antibodies. Serologic assays alone cannot

answer the question, Does this patient have Lyme disease? Tests may be positive due to

exposure to cross-reacting antigens or to the persistence of B. burgdorferi antibodies

after a previous infection unrelated to a current illness. The clinical relevance of a test

result reporting antibodies to B. burgdorferi depends on the pretest probability that the

patient has Lyme disease, as well as on the sensitivity and specificity of the assay used.

When these characteristics are understood and taken into account, serology is the most

useful tool for assessing exposure to B. burgdorferi in all stages of Lyme disease after

the first 2 to 3 weeks of infection (Aguero-Rosenfeld et al., 2005; Johnson, 2006;

Wilske et al., 2007a).

Lyme Borreliosis in Europe and North America: Epidemiology and Clinical Practice, First Edition.Edited by Sunil K. Sood� 2011 Wiley-Blackwell. Published 2011 by John Wiley & Sons, Inc.

The findings and conclusions in this report are those of the authors and do not necessarily represent the official

position of the Centers for Disease Control and Prevention (CDC). The use of trade names is for information

purposes only and does not constitute endorsement by the CDC.

185

TWO-TIERED SEROLOGY: A FIRST STANDARD FOR SERODIAGNOSISIN THE UNITED STATES

After B. burgdorferi sensu strictowas detected in ticks of the genus Ixodes and cultivated in

vitro in 1981, the first serologic assays for Lyme disease were developed (Burgdorfer

et al., 1982; Barbour, 1984; Russell et al., 1984). The number of assays in use proliferated,

but the performance characteristics of many tests were inadequate to serve the needs of

either clinicians or epidemiologists. These tests lacked specificity, and the sensitivity and

reproducibility of various tests were poorly understood (Magnarelli, 1989; Bakken

et al., 1997). Few laboratories had evaluated these tests using serum samples from patients

with clinically well-characterized Lyme disease, or from patients with culture-confirmed

B. burgdorferi infection or seroconversion demonstrated in serially drawn specimens

(ASTPHLD, 1991).

In the early 1990s efforts were made to ameliorate this unacceptable testing situation by

investigators in clinical reference centers, public health agencies, and private industry. A

two-test approach for Lyme disease serology had been adopted by several academic

laboratories serving major clinical referral practices. When these laboratories participated

in a multicenter evaluation of serodiagnostic assays by testing blind-coded samples from

clinically well-characterized Lyme disease patients and healthy blood donors, the three

reference centers that used Western immunoblotting to supplement their enzyme immu-

noassay (EIA) results performed much better than the three that used EIAs alone (Craven

et al., 1996). These results provided part of the foundation for the initial standardization of

serology. Criteria for the scoring and interpretation of both IgM and IgG immunoblots were

derived and evaluated (Dressler et al., 1993; Engstrom et al., 1995). In late 1994 the

Association of State and Territorial Public Health Laboratory Directors (ASTPHLD), the

Centers for Disease Control and Prevention (CDC), and other agencies recommended that a

two-step approach (commonly called “two-tiered serology”) be adopted for testing serum

specimens (ASTPHLD, 1995; CDC, 1995). The first commercial immunoblot devices (kits)

for Lyme disease serology were cleared by the US Food and Drug Administration (FDA) in

1996 (www.fda.gov, product code LSR).

The public health community advocated two-tiered testing not only as a means to

improve care of individual patients and epidemiologic surveillance for Lyme disease but

also to establish a standard against which new, potentially improved, serologic methods

could be evaluated. The FDA adopted the position that before new tests could be cleared as

alternatives to two-tiered testing, test performance should be equal to or better than the

recommended two-test procedures (Brown et al., 1999)

The essential features of two-tiered serology are illustrated in Figure 10.1. The first tier

consists of a sensitive initial serologic test or tests that detect class-specific antibodies

(IgM and IgG, either together or separately) by an EIA or, less commonly, an immuno-

fluorescent assay (IFA). If the first-tier assay result is negative, the serum is reported to be

negative for antibodies to B. burgdorferi and is not tested further. Serum samples that are

found to be reactive (“positive”) or indeterminate (called “equivocal” or “borderline” in

some tests) are tested by supplemental standardized Western immunoblotting. If an IgG

immunoblot is scored as positive, two-tiered testing is reported as positive. If an IgM

immunoblot is scored as positive, Lyme disease serology is reported as positive with the

caveat that this finding is clinically relevant only in early disease, that is, in the first month

of illness (CDC, 1995).

186 SERODIAGNOSIS OF LYME BORRELIOSIS

It is important to appreciate that the first and second tier tests are not independent

indicators of exposure to B. burgdorferi (Wormser et al., 2000). A Western blot cannot

appropriately be said to “confirm” the results of a first tier test. The initial and supplementary

tests usually are constructed with essentially the same antigens—whole-cell antigens of

bacteria grown in culture—but they are processed differently. EIAs provide an estimate of

the magnitude of the IgG/IgM humoral antibody response to all of the antigens that are

expressed under the culture conditions used to produce the antigen. EIA results are objective

and quantitative. They can be correlatedwith antibody titers. Immunoblotting techniques, in

contrast, separate the many bacterial antigens spatially on a solid support so that the

specificity and complexity of the antibody responses are revealed. The rationale for

determining IgM and IgG antibody profiles is to learn whether the antibodies recognize

proteins of B. burgdorferi that have been established to be more predictive of Lyme disease

than other components of the bacteria. Many antigens have similarities to those of

other organisms, such as proteins involved in motility (e.g., flagellin) and responses to

Two-Tiered Serology

(Immunoblotting Conditionally Supplements EIAs)

Tier 1: IgG or IgM EIAs (combined or separate)

Tier 2: IgG and/or IgM Immunoblots (separate)

Positive or Indeterminate Negative

Reported as negative; 2-tiered protocol complete

Blot Negative

Reported as negative

Blot Positive

IgG positive reported as positive IgM positive clinically relevant only in early disease of less than about 1 month duration

Figure 10.1 Two-tiered serology for Lyme disease. The first tier is shown as an EIA. Immunoflu-

orescent assays (IFAs)may be substituted, but they are uncommonly used since they require a skilled

microscopist and generally cannot be scored objectively. When a serologic result is negative and

early Lyme disease is still suspected on clinical grounds, a second samplemay be tested 2 to 3weeks

later, preferably paired with the original specimen.

TWO-TIERED SEROLOGY: A FIRST STANDARD FOR SERODIAGNOSIS 187

stress (e.g., “heat shock” proteins). Recognition of one or more antigens from this set by

serum antibodies is not necessarily indicative of exposure to B. burgdorferi, although these

reactions contribute to the signal strengthmeasured in anEIA. Immunoblots usually contain

variable amounts of each protein (reflecting the relative expression of each of them in

culture) and are qualitative, not quantitative tests.

The protein “bands” selected for scoring were chosen from all of the immunogenic

proteins detected in immunoblots. The frequency of recognition of each protein was

compared between serum samples from clinically characterized Lyme disease patients

and samples from healthy donors (Engstrom et al., 1995) or patients with other neurologic,

rheumatologic, or infectious illnesses (Dressler et al., 1993). Proteins that were more

frequently recognized by serum samples from Lyme disease patients compared with

controls, as judged by tests of statistical significance, were ranked and evaluated in

combinations. Early in infection, IgM responses predominate and relatively fewer

antibodies of any class are detectable compared with infections of longer duration. IgM

blots are scored as positive if at least 2 out of 3 diagnostically significant bands (that were

determined by chi-square analysis) are present (Engstrom et al., 1995). For IgG, the

number of the most common bands selected for scoring was chosen by receiver operating

characteristic (ROC) analysis, a technique that maximizes the area under a graph of test

sensitivity versus (1-specificity). IgG blots are scored as positive if at least 5 out of 10

designated bands are present (Dressler et al., 1993). Examples of immunoblots are shown

in Figure 10.2.

An immunoblot is scored with reference to a calibration control (positive control) that

identifies the positions of diagnostically significant bands. In commercial blots, this

calibrator is usually a polyclonal serum sample that has a well-characterized pattern of

reactivity, often determined by use of monoclonal antibodies developed to identify specific

proteins. Monoclonal antibodies themselves may be used as calibrators.

In addition a control for reaction strength (sometimes called a “weak-positive” control)

is necessary to aid in deciding whether a band has sufficient color density to be scored. This

decision is a major source of difficulty in the interpretation of immunoblots. The problem is

that there is an inherent low level of “stickiness” between proteins, a background against

which significant antibody–antigen interactions must be judged. In general, IgM antibodies

are more nonspecifically “sticky” than IgG antibodies, owing to their pentameric (rather

thanmonomeric) structure. Faint bands, particularly in IgMblots, may not be diagnostically

significant even for so-called specific antigens. Unfortunately, data describing thevalidation

of the “weak-positive” controls in commercial tests are not published. There is unease

among specialists in some academic and government reference laboratories that it is not

uncommon for samples to show “significant” IgM bands of an intensity greater than the

weak-positive control that probably reflect nonspecific reactivity. Manufacturers should

make an effort to demonstrate in the scientific literature that their reaction strength controls

produce valid results. If health care providers adhere to the recommendation to demonstrate

that antibodies are present at a positive or indeterminate level by a first-tier test before

ordering an immunoblot, the risk of an erroneously positive serology based on scoring faint

bands is reduced, but not eliminated.

Assays conditions influence whether nonspecific stickiness will result in a detectable

band. These conditions include such variables as the concentrations of salts, detergent, and

detecting reagents; assay temperature; and lengths of time allowed for antibodies to interact

with antigens and for development of blot signal (usually color). Since assay designs differ

among commercial products and “in-house” tests, awell-validated reaction strength control


is test-specific.1 Even when a reaction strength control is valid, the scoring of blots involves

some subjectivity. The knowledge and experience of the reader may influence the result.

Densitometers are usedwith some blot kits in attempt tominimize this problem, but operator

judgment is still involved.

A negative control is required for each group of immunoblot strips run at the same time.

This control serum may have some faint bands, for the reasons above, but should give a

negative result when scored by the blot interpretation criteria.

1 The rate of color development by the reaction strength controlmust be linear over the same time frame as the bands

in the test serum sample. Difficulties can arise when the control develops full color before the patient’s sample. The

ratio of signal strength in a patient’s sample comparedwith the reaction strength controlwill go upwith length of time

allowed for color development. If development time is excessive, the outcome will be a false-positive test result.

Figure 10.2 Examples of conventional IgM and IgG immunoblots. (Left panel) IgM blots; (right

panel) IgG blots. P, positive control serum;N, negative control serum; C, calibration control (weak

positive control); kDa, molecular mass in kilodaltons. Calibration control bands are typically light

and reproduce poorly, so they have been digitally enhanced to be more visible. The arrows

identify the bands that are recommended for scoring when a patient has been exposed to B.

burgdorferi in the United States. Two additional bands are labeled in the IgG blot (outer surface

proteins A and B at 31 and 34 kDa, respectively). IgMblots profiles are for a patient with acute EM

(lane 1) and for the same patient at convalescence (lane 2). IgG lanes 1 to 8 show the profiles of 8

patients with later manifestations of Lyme borreliosis. The antigen was B. burgdorferi sensu

stricto strain B31.


Most serologic assays currently in use (first- and second-step assays) employ antigen

preparations obtained from B. burgdorferi cultured in vitro that do not contain all antigens

presented to the immune system during active infection. During mammalian infection,

borreliae expressmany antigens that are absent (or present at only low levels) in cells grown in

laboratory cultures. Some newer serologic assays use antigens that are expressed preferen-

tially in vivo. They are produced by recombinant DNA techniques or other synthetic means.

Performance Characteristics of First-Tier Tests

Types of First-Tier Tests Available Commercially

Although 52 different first-tier tests developed by 21 companies were approved by the FDA

between 1987 and 2009, many of the cleared assays are no longer marketed (http://www.

accessdata.fda.gov/scripts/cdrh/cfdocs/cfPMN/pmn.cfm, product code LSR, accessed

3/26/2010). Two-thirds (35/52) of these tests were approved before the era of two-tiered

serology (1994), when whole-cell antigen EIAs and IFAs were designed to stand alone.

Many were inadequately sensitive in order to achieve a modicum of specificity (Bakken

et al., 1997). Currently a relatively small number of whole-cell antigen EIAs that have been

tuned to optimize sensitivity are in widespread commercial use, and unlike some early tests,

they are suitable for use in a two-tiered testing scheme. FDA-authorized first-tier tests made

by five companies were used by about three-quarters of the nearly 400 laboratories

that participated in the 2007 College of American Pathologists proficiency surveys; the

EIA of one company was used for more than 40% of the results reported (CAP, 2007).

First-tiered tests based on synthetic or recombinant antigens, rather than lysates of whole

cells, recently have been approved by the FDA. They are based on an immunodominant

antigen, VlsE. VlsE is expressed well by borreliae in mammalian hosts but is produced at a

very low level in the culture medium usually used for growth of organisms in the laboratory.

In consequence this diagnostically useful antigen is effectively lacking in traditional two-

tiered assays. The gene encoding the VlsE protein undergoes rapid recombination in vivo,

resulting in a large repertoire of antigenic variants, but maintains certain invariant regions

(Zhang et al., 1997). One small peptide of VlsE, a 25-amino acid portion of the sixth

invariant region (IR6) plus a cysteine to aid in assay design, has proven to be remarkably

useful as a diagnostic antigen (Liang et al., 1999). The prototype test is called theC6 assay.A

version of the C6 assay designed and produced by Immunetics, Inc. was authorized by the

FDA for commercial use in 2001. In this assay the peptide reproduces the IR6 found in

B. burgdorferi sensu stricto strain B31.A diagnostic assay containing entire VlsEmolecules

expressed as recombinant proteins from both B. burgdorferi and Borrelia garinii, a species

of Lyme disease Borrelia found only in Eurasia, became available as a first-tiered test in

2007 (Diasorin Liaison, www.fda.gov).

Sensitivity of First-Tier Tests by Stage of Lyme Disease

The antibody response to B. burgdorferi develops slowly over the first few weeks after the

spirochete is introduced into the body. Since ticks bites often are not noticed, the course of

development of serum immune responses generally is studied as a function of time after the

first appearance of erythema migrans (EM) (Aguero-Rosenfeld et al., 1993, 1996). Patients

with EM are often seronegative at the time of presentation (Table 10.1). The probability of


seroreactivity is low during the first week after EM becomes visible. It increases with

duration of EM and with the development of signs of disseminated disease. By convales-

cence, however, up to 80% of treated EM patients are seropositive. The optimal time for

detecting antibodies in treated EMpatients is 8 to 14 days after the start of antibiotic therapy

(Engstrom et al., 1995; Aguero-Rosenfeld et al., 1996). Most of the treated patients who

failed to seroconvert at convalescence in the studies in Table 10.1 were those who had a

single localized EM lesion without evidence of disseminated infection.

From about 2 to 6 weeks after initial infection, antibodies rise in titer, recognize an

increasing number of borrelial antigens, and switch classes from predominantly IgM

responses to IgG. Because the antibody responses are evolving during the first weeks of

infection, some patientswith clinical presentations compatiblewith early Lymediseasemay

not yet be seropositive. In this situation serologic evidence of infection is best obtained by

testing paired samples that are obtained several weeks apart. If paired samples are tested in

parallel, increasing titers can support the clinical diagnosis of Lyme disease, but unfor-

tunately this serologic tool is seldom used in routine clinical practice.

The evolution of the immune responses during early infection is illustrated by the

serologic findings in patients with early neurologic disease. In a study of patients with facial

paralysis, 87%had diagnostic levels of IgM antibodies, 66%were IgG positive, and all were

seropositive for at least one antibody class (Peltomaa et al., 2004) (Table 10.1). This profile

of antibody reactivity by class (i.e., greater frequency of positive IgM responses than IgG,

with many people seropositive for both classes) also is seen in patients with other

manifestations of early neurologic disease, typically meningitis and/or radiculoneuritis

(Roux et al., 2007). Fewer data are available for less commonmanifestations of infection of

the nervous system such as parenchymal inflammation of the brain or spinal cord or

radiculopathy. Physicians should be aware that patients with early neurologic Lyme disease

commonly have a history of recent EM. Lyme facial paralysis, for example, was associated

with EM in 72% to 87% of patients, depending on the study (Peltomaa et al., 2004).

Another possible presentation of Lyme disease during the early weeks of infection is

carditis, whichmanifests primarily as atrioventricular block. Themedian time from onset of

EM to carditis is about 3 weeks (range 4–83 days) (Steere et al., 1980). Lyme carditis is

uncommon, occurring in 0.5% to 2% of Lyme disease patients in the United States. It

appears to have even lower incidence in Europe (Haddad and Nadelman, 2003). In

consequence few serum samples from these patients have been available in retrospective

or prospective evaluations of serologic tests. The vast majority of carditis cases are reported

to be seropositive at presentation (Wormser et al., 2006). Some patients develop cardiac

manifestations after a short duration of infection and may not yet be seropositive, under-

scoring the diagnostic importance of finding other signs and symptoms of Lyme disease.

Lyme carditis typically occurs in patients with previous or concurrent EM (>80%) or

sometimes early neurologic Lyme disease.

By the time patients develop later manifestations of Lyme disease, they are almost

universally seropositive (Table 10.1). Numerous studies with robust samples sizes have

been published about the immune responses in Lyme arthritis. Patients with Lyme

arthritis typically have high IgG titers, higher than those seen in any of the various

manifestations of Lyme disease, and waning IgM responses (Dressler et al., 1993; Akin et

al., 1999; Kannian et al., 2007). Although an IgM response to a protein of 34 kDa (known

as outer surface protein B or OspB) has been observed to develop late in infection in

patients with prolonged disease, this new response occurs in the context of a robust IgG

response to a large number of antigens. IgM responses in the absence of diagnostic levels


TABLE10.1

Sensitivity

offirst-tiertestsforantibodiesto

B.burgdorferiin

U.S.patientsby

clinicalmanifestationof

infection

Seropositivity(%

)

Acute

Erythem

aMigrans

(Daysfrom

Onset)

Acute

StageofOther

Manifestations

Convalescent

Reference

�7Days

>7Days

AtPresentation

Post-treatment

Erythem

amigrans

WCELISA

1,IgG

þIgM

aAguero-Rosenfeld

etal.,1996

10

71

84c

WCELISA

2,IgG

þIgM

bJohnsonet

al.,2006;

Baconet

al.,2003

31

89

95d

C6kinetic

ELISA,IgGb

Johnsonet

al.,2006;

Baconet

al.,2003

30

52

75d

Early

neurologic

disease

WCELISA

2,IgG

þIgM

eJohnsonet

al.,2006

100

WCELISA

2,IgG

þIgM

eJohnsonet

al.,2006

100

WCELISA

3,IgM

fPeltomaa

etal.,2004

87

WCELISA

3,IgGf

Peltomaa

etal.,2004

66

WCELISA

3,IgM

orIgGf

Peltomaa

etal.,2004

100

C6ELISA,IgGf

Peltomaa

etal.,2004

100

192

Lymearthritis

WCELISA

3,IgM

gDressleret

al.,1993

16

WCELISA

3,IgGg

Dressleret

al.,1993

100

WCELISA

2,IgG

þIgM

hJohnsonet

al.,2006

97

WCELISA

2,IgG

þIgM

hJohnsonet

al.,2006

100

WCELISA

3,IgGi

Kannianet

al.,2007

100

WCELISA

3,IgGi

Kannianet

al.,2007

100

C6ELISA,IgGi

Kannianet

al.,2007

100

C6ELISA,IgGi

Kannianet

al.,2007

93

Lateneurologic

disease

WCELISA

3,IgM

jDressleret

al.,1993

4

WCELISA

3,IgGj

Dressleret

al.,1993

84

WCELISA

2,IgG

þIgM

kJohnsonet

al.,2006

100

aWC¼whole

cellantigens.All46EM

casesconfirm

edbyculture

ofB.burgdorferi.Calculatedfrom

Aguero-Rosenfeld

etal.,1996.

bEM

(n¼80)confirm

edbyculture

(n¼38)orbyevidence

ofdisseminated

infection(n¼35).Calculatedfrom

Baconet

al.,2003;Johnson,2006,andJohnsonunpublished.

cObtained

8–14daysafterthestartofantibiotictherapy.

dObtained

2–4weeksafterthestartofantibiotictherapy.

eAcuteandconvalescentsamplesnotpaired.Acutesamplesfrom

patientswithmeningitis(10/15),facialparalysis(8/15),andradiculoneuropathy(3/15);convalescentsamplesfrom

patientswithmeningitis(5/11),facial

paralysis(5/11),andradiculoneuropathy(2/11).

f Facialparalysis,commonly

withrecentEM

(34/47);samplesobtained

within

6weeksofonsetofparalysis.

gLymearthritis(n¼25).

hAcute

(n¼33)andconvalescent(n¼24)samplesnotpaired.

i Acute

samples(n¼23)from

patientswithantibiotic-responsivearthritis;post-treatmentsamples(n

¼41)from

patientswithantibiotic-refractory

arthritis.

j Lateneurologic

disease

(n¼25)manifestedas

encephalopathyorpolyneuropathy;abouthalfhad

received

antibiotictherapypriorto

adiagnosisofneurologic

Lymedisease.

kLateneurologic

disease

manifestedas

encephalopathy(9/11)andpolyneuropathy(3/11).

193

of IgG antibodies should not be used to support the diagnosis of any manifestation of late

Lyme disease.

Late neurologic Lyme disease, presenting as encephalomyelitis, peripheral neuropathy,

or encephalopathy, is rare (Wormser et al., 2006; Halperin et al., 2007). It has been

speculated that late neuroborreliosis has become rarer in recent years due to earlier

diagnosis and treatment, preventing progression to late-stage manifestations. Serum IgG

antibodies have been consistently found in the patients who have been available for study. In

the 2003 study summarized in Table 10.1, 100% of 11 late neurologic Lyme disease patients

were seropositive (Bacon et al., 2003). These patients were classified as having late

neurologic Lyme disease not only because of their neurologic findings but also because

of antecedent other clinical manifestations of Lyme disease that were well documented.

This demanding standard for admission to the study was necessary, since the signs of late

neurologic Lyme disease, particularly encephalopathy, are not unique to infection by

B. burgdorferi.

The serologic characteristics of patients who experience ongoing pain, fatigue, and/or

memory difficulties after appropriate courses of antibiotic therapy for Lyme disease

are beyond the scope of this review. This condition has been variously called post–Lyme

disease syndrome, post-treatment Lyme disease syndrome, or “chronic Lyme disease.”

The preponderance of evidence indicates that these patients are no longer infected with

B. burgdorferi (Feder et al., 2007). Serologic testing of patients with post–Lyme disease

symptoms may be either positive or negative, depending on the clinical stage of

Lyme disease when antibiotic therapy was started and the length of time that has passed

since the infection was treated (Klempner et al., 2001).

Inadequate Specificity of First-Tier Tests Based on Whole-Cell Antigens

Although EIAs performed as first-tier serologic assays have excellent sensitivity after

the first weeks of infection, alone they usually have inadequate specificity. Inadequate

specificity is a characteristic of EIAs based on whole-cell antigens of B. burgdorferi, which

are derived from cells grown in vitro. Whole-cell EIAs currently in common use have

specificities of around 95% when used to evaluate healthy blood donors from North

American areas that are not endemic for Lyme disease (Johnson, 2006). When healthy

donors from endemic areas are tested, higher rates of reaction in EIAs are seen, owing to the

persistence of antibodies years after active Lyme disease (Kalish et al., 2001) and

seroconversion of exposed individuals who do not develop clinical illness (asymptomatic

seroconversion rate of around 10%) (Steere et al., 1998). Since these latter positive

reactions are true anti–B. burgdorferi antibodies, the specificities of EIAs are typically

determined with samples from non-endemic areas.

High rates of false-positiveEIA results (up to about 55%) arewell documented in patients

with other spirochetal illnesses, particularly syphilis, leptospirosis, and tick-borne relapsing

fever, where cross-reactivity would be expected on biologic grounds (Johnson, 2006).

Positive EIAs may occur with serum from patients with periodontal disease when

treponemes are present in gingival pockets, although data are sparse. Samples from patients

with other spirochetal infections are commonly tested by developers of serologic assays.

Such samples provide a rigorous challenge of an assay’s specificity since common antigenic

motifs among spirochetes are well known. Furthermore some Lyme disease antibody

assays have included other spirochetal antigens in their format as a means of adsorbing


cross-reacting antibodies. Although the results of testing serum samples from patients with

other spirochetal diseases are instructive to microbiologists, they are less pertinent to

physicians who can distinguish these infections clinically from Lyme disease, with the

possible exception of tertiary syphilis.

In patients with rheumatologic, neurologic, or other conditions within the differential

diagnosis of Lyme disease, false-positive rates of around 10% have been observed,

depending on the population (Johnson, 2006). This generalization applies to patients with

antinuclear antibodies, rheumatoid factor, clinical rheumatoid arthritis, or multiple scle-

rosis. Studies are still needed of a larger number and wider array of patients with illness that

may be difficult to distinguish clinically from Lyme disease.

The issue of false-positive Lyme disease serology reported in patients with human

granulocytic anaplasmosis (HGA) is very difficult to study. Investigators usually cannot rule

out coinfection or prior infections withB. burgdorferi (Wormser et al., 1997). Other reasons

reported to give nonspecific reactivity inLyme disease EIAs are polyclonalB cell activation,

Epstein Barr virus (EBV) infections, or malaria (Kaell et al., 1993; Magnarelli, 1995;

Burkot et al., 1997). There also have been reports of nonspecific reactions in tropical serum

samples (Burkot et al., 1997).

Serum from people who received the recombinant OspA Lyme disease vaccine

(approved by the FDA in 1999, but removed from the market in 2002) will react in

whole-cell EIAs (Aguero-Rosenfeld et al., 1999). Although this potential source of false

positivity does not affect a large percentage of the population, it is appropriate to inquire

whether a patient received the OspA vaccine. Two-tiered testing will generally

be negative, however, since the immunoblot reactivity of serum from vaccinees is

distinctive and does not fulfill either the IgG or the IgM blot criteria.

The specificities of EIAs developed with a synthetic, immunodominant peptide of the

VlsE molecule (C6 peptide) or with whole recombinant VlsE are superior to EIAs based on

whole-cell antigens. These assays usually achieve specificities of 98% or higher, depending

on the test and how the cutoff value for a diagnostically significant reaction was set (Liang

et al., 1999; Bacon et al., 2003; Kannian et al., 2007).

Conditional Immunoblotting to Improve the Specificity of Serology

The specificity of serology is significantly improved when immunoblotting is added to

the testing protocol. Blotting should be performed “conditionally,” that is, depending on

whether there is some evidence of the presence of antibodies to B. burgdorferi as

determined by a first-tier test.When a first-tier test is positive or indeterminate, assessment

of the reactivity profile of the antibody response on immunoblot permits discrimination

between true and false positives. The utility of conditional blotting is illustrated by the

retrospective study summarized in Table 10.2 (Bacon et al., 2003; Johnson, 2006).

Whereas the specificity of the EIAwas 96% for healthy blood donors, two-tiered testing

improved specificity to 100%. For a panel of samples from patients with diverse other

illnesses, both infectious and noninfectious, conditional blotting increased specificity

from 85% to 98% (Table 10.2a).

Crude estimates of the number of serologic tests for Lyme disease performed annually in

the United States range between 1 and 2 million assays. If these estimates are correct, each

1% improvement in test specificity saves 10,000 to 20,000 false-positive results. To put this

number in context, in recent years about 20,000 cases of Lyme disease have been reported


annually to the CDC as part of the US national system for surveillance of notifiable diseases

(CDC, 1997, 2007).

Adequate specificity comes at a cost. A fundamental characteristic of diagnostic testing

is that there is a trade-off between specificity and sensitivity. Serologic testing for antibodies

to B. burgdorferi is no exception. The trade-off has the greatest consequence in early

borreliosis, when antibody concentrations are still low. As illustrated in Table 10.2b, two-

tiered testing of serum from patients with EM ismuch less sensitive than use of awhole-cell

lysate EIA. EIAs detect the sum of interactions of antibodies with dozens of antigens,

whereas blots are scoredwith respect to 3 (for IgM) and 10 (for IgG) specific proteins. In late

Lyme borreliosis, such as Lyme arthritis or late neurologic disease, the immune system has

had time to develop antibodies to more antigens and in higher concentrations, so the

difference in sensitivity between EIAs and two-tiered results greatly diminishes or

disappears.

The sensitivity of serologic testing of patients with EM is inadequate to be clinically

useful. In acute EM, sensitivity is commonly less than 40% (Table 10.2b). In patients with a

single EM and no symptoms of disseminated infection (e.g., headache, stiff neck, arthral-

gias), the sensitivity of two-tiered testing is only about 25% (Bacon et al., 2003). In patients

with symptoms of disseminated infection or multiple EMs, sensitivity doubles to about

T A B L E 10.2 Sensitivity and specificity of two-tiered testing

a. Specificity of serology is improved when first-tier testing is supplemented by conditional

immunoblotting.a

Specificity (%)

Non-Lyme disease n EIA Two-Tier

Healthy U.S. donors 257 96 100

Conditions other thanLymedisease, total 292 85 98

Rheumatoid arthritis and/or

rheumatoid factor positive

109 94 99

Anti-nuclear antibody positive 116 93 98

Other spirochetal diseases 67 58 97

b. Sensitivity of two-tiered testing is good for later stages of disease, but not for

erythema migrans (EM).a

Sensitivity (%)

Stage of Lyme disease n EIA Two-Tier

EM, acute 80 60 38

EM, convalescent 106 91 67

Early neurologic disease, acute 15 100 87

Lyme arthritis 33 97 97

Late neurologic disease 11 100 100

aConcepts illustrated with data from Johnson, 2006 and Bacon et al., 2003.


50%. Lower antibody detection rates have been reported in culture-positive European

patients compared with patients from the United States, possibly due to differences in the

causative agents (mainly Borrelia afzelii versus B. burgdorferi) and the fact that multiple

EMs occur more often in the United States (Strle et al., 1999). It is appropriate to treat EM

patients with antibiotics based on their clinical presentation and history of probable

exposure to infected ticks in an endemic area. “Treat but don’t test” has long been the

position of an expert panel of the American College of Physicians that estimated that

the pretest probability of Lyme disease in this circumstance is greater than 0.8 (Tugwell

et al., 1997).

After the first weeks of infection and coincident with the development of signs of

disseminated disease, the sensitivity of two-tiered testing is good (Table 10.2b). In early

neurologic Lyme disease the sensitivity of two-tiered testing approaches 90%. Some

patients with early neurologic Lyme disease may have an illness of 5 to 6 weeks’ duration

and yet not have an IgG response that meets blot criteria; they may, however, fulfill IgM

blot criteria instead. It has been suggested that IgM testing may be useful until about 6

weeks after onset of Lyme borreliosis (Kannian et al., 2007), about 2weeks longer than the

period recognized in the 1995 recommendations (ASTPHLD, 1995; CDC, 1995). As

discussed above, if serology is negative at the time that a patient is initially examined, it

may be useful to test a second sample a few weeks later if Lyme disease is still suspected.

In patients with Lyme arthritis or with late neurologic disease, the sensitivity of two-tiered

testing is close to 100%.

Strengths and Weaknesses of Two-Tiered Testing

This diagnostic algorithm has been evaluated both retrospectively (see above) and

prospectively (Steere et al., 2008). It has high specificity for determination of IgG antibodies

to B. burgdorferi. Experienced laboratories with good quality control and quality assurance

programs obtain consistent results (Bacon et al., 2003; Kannian et al., 2007; CAP, 2007).

The specificity of IgM testing is not as good (Engstrom et al., 1995; Johnson et al., 1996),

which accounts for the recommendation that IgM testing should be performed only in the

first month of illness (ASTPHLD, 1995). Some investigators in reference laboratories

believe that two-tiered IgM testing using current methods should not be done, both for

reasons of specificity and because the duration of illness is often unclear or at least

undocumented by good clinical information in requests for testing (unpublished Banbury

conference summary, The Laboratory Diagnosis of Lyme Disease, September 2007).

Recently an IgG immunoblot with VlsE band was described that eliminates the need for

IgM testing (Branda et al., 2010). It provides superior sensitivity in acute neuroborreliosis

and carditis, while maintaining high specificity.

Two-tiered testing is insensitive in acute EM and not useful in clinical practice.

Sensitivity of standard two-tiered testing in early neurologic disease is less than ideal

(Table 10.2b), although the new IgG algorithm incorporating VlsE increased sensitivity

from 63% to 96% (Branda et al., 2010). The recommendation to obtain a second blood

sample a few weeks after the initial one to seek evidence of seroconversion is inconvenient

and costly, and causes delay in diagnosis. Furthermore commercial testing laboratories are

generally not set up to save initial specimens. To obtain the most reliable results, initial and

subsequent samples should be tested at the same time, but this is practical only in research or

academic settings.


The antibody responses to B. burgdorferi increase in magnitude and diversity with

duration of infection, as discussed earlier. In consequence immunoblotting band profiles

increase in intensity and complexitywith duration of infection aswell. These characteristics

can be diagnostically useful in specialized circumstances, such as when the results of

serially drawn blood samples are available for the atypical patient who remains unwell after

a standard course of antibiotic therapy. Expansion of the number and intensity of

immunoblot bands is compatible with ongoing infection. In general, though, reporting

of specific banding patterns (including those not considered to be “significant bands”) is

counterproductive. This practice gives rise to the erroneous concept of a “partially positive”

or “nearly positive” blot, which causes diagnostic confusion particularly when a patient is

tested despite a low pretest probability of Lyme disease (e.g., lacks objectives signs of

borreliosis). Clinical laboratories sometimes prominently report banding patterns and give

inadequate attention to the interpretation of these results. When this is done, health care

providers may be confused and develop the mistaken idea that the presence of any number

of bands is a significant finding. Specific bands, if they are reported at all, should be de-

emphasized in the report.

Immunoblots are complex, technically demanding, and difficult to standardize. Some

laboratories are inexperienced in properly using blot color development cutoff controls.

The Clinical Laboratory Standards Institute (formerly known as National Committee for

Clinical Laboratory Standards, NCCLS) notes that “The erroneous scoring of a faint band

is a common reason for false-positive readings. . .” (NCCLS, 2000). IgM results are more

affected by this problem than IgG blots (Johnson et al., 1996). Only 2 of 3 specified bands

are required for an IgM blot to be reported as positive, whereas 5 of 10 bands are necessary

for an IgGblot to be positive by the recommended blot interpretation criteria (CDC, 1995).

A single erroneously scored faint band will affect IgM results more readily than IgG

results, especially since about half of the population have antibodies that react with one of

the IgM bands (FlaB, a protein of 41 kDa), regardless of Lyme disease status (Aguero-

Rosenfeld et al., 1996). Densitometers may be used to assist with judging whether a given

band meets the threshold for being reportable, but they will not help when underlying test

design and color development problems are present.

In sum, two-tiered testing brought order to a chaotic testing environment in the early

1990s and improved the performance of serology for Lyme disease. It has worked well for

determination of IgG antibodies after the first weeks of infection, despite its complexity, but

works less well for determination of the IgM response. It also defines a standard against

which new testing approaches can be judged.

HETEROGENEITY OF B. burgdorferi SENSU LATO IN EUROPE:IMPLICATIONS FOR SERODIAGNOSIS

The diversity of Lyme disease spirochetes, as well as the diversity and variability of their

protein-encoding genes, impact laboratory diagnosis of Lyme borreliosis in Europe.

Heterogeneity of Causative Strains

In contrast to the United States where only one species, B. burgdorferi sensu stricto, causes

Lyme disease, the human pathogenic strains in Europe belong to several different species


that vary in their distribution by geographic area. These species are B. burgdorferi sensu

stricto, Borrelia afzelii, Borrelia garinii (various serotypes have been described for

B. garinii [Wilske et al., 1993], and serotype 4 has been named B. bavariensis [Margos

et al., 2009]), and Borrelia spielmanii (former genospecies A14S) (Wang et al., 1999;

Richter et al., 2006). Borrelia valaisiana and Borrelia lusitaniae have been associated

anecdotally with Lyme disease in some parts of Europe, particularly B. lusitaniae in

Portugal. The major species have been associated with different clinical manifestations of

the disease. B. afzelii has a high prevalence among isolates from human skin, especially

those from patients with acrodermatitis chronica atrophicans (ACA), a chronic skin

condition that has not been described in theUnited States (Canica et al., 1993; Ohlenbusch

et al., 1996; Wilske et al., 1996b; Ruzic-Sabljic et al., 2000; Fingerle et al., 2008)

(Table 10.3a). Isolates from cerebrospinal fluid most often belong to B. garinii (Wilske

et al., 1993, 1996a,b; Eiffert et al., 1995; Ruzic-Sabljic et al., 2001; Fingerle et al., 2008)

(Table 10.3b). Reports differ among authors on whether B. burgdorferi sensu stricto is

preferentially associated with Lyme arthritis (Eiffert et al., 1998; Vasiliu et al., 1998;

Jaulhac et al., 2000). With the exception of a study from France (Jaulhac et al., 2000),

borreliae detected by PCR in synovial fluids from European patients with Lyme arthritis

are highly heterogeneous. This high heterogeneity was confirmed by culture of three

species of Borrelia from synovial fluids of Lyme arthritis patients (Fingerle et al., 2008)

(Table 10.3c). To date, B. spielmanii has been isolated only from skin biopsy specimens

(Fingerle et al., 2008).

T A B L E 10.3 Borrelia species in skin, CSF, or synovial fluid of EuropeanLyme borreliosis patients

a. Borrelia species isolated from skin

Reference Number B. burgdorferi s.s. B. afzelii B. garinii B. spielmanii

Ruzic-Sabeljic et al., 2000 87 1% 85% 14% not done

Fingerle et al., 2008 160 6% 67% 24% 2.5%

Wilske et al., 1996b 68 6% 84% 10% not done

b. Borrelia species detected in CSF

Reference Method Number B. burgdorferi s.s. B. afzelii B. garinii

Eiffert et al., 1995 PCR 12 0% 33% 67%

Ruzic-Sabeljic el al., 2001 culture 40 2.5% 35% 62.5%

Wilske et al., 1996a culture 37 11% 24% 65%

c. Borrelia species detected in synovial fluid

Reference Method Number B. burgdorferi s.s. B. afzelii B. garinii

Fingerle et al., 2008 culture 6 2 2 2

Eiffert et al., 1998 PCR 7 3 1 3

Jaulhac et al., 2000 PCR 10 9 0 1

Vasliu et al., 1998 PCR 15 4 5 6

HETEROGENEITY OF B. burgdorferi SENSU LATO IN EUROPE 199

Molecular Heterogeneity of Immunodominant Proteins andImplications for Serodiagnosis

Amino acid sequence identities among major immunodominant proteins (DbpA, VlsE,

OspC, OspA, BmpA, p83/100, p58, and flagellin) from the three main human pathogenic

species range from 40–44% to 96–97% (Table 10.4). The most heterogeneous protein

is DbpA (decorin binding protein A). Six distinct DbpA groups have been described

(Schulte-Spechtel et al., 2006; Fingerle et al., 2008). Group I is comprised of B. burgdorferi

s.s., and group II of B. afzelii. B. garinii, the most heterogeneous species, is divided into

groups III and IV, whereas B. spielmanii strains form groups V and VI despite being

homogeneous in OspA. OspA is not only heterogeneous among species but also rather

heterogeneous amongB. garinii strains (Wilske et al., 1996b). OspC has conserved epitopes

primarily recognized by IgM antibodies, but also type-specific ones especially recognized

by IgG antibodies (Wilske et al., 1996b). These factors make OspC a poor antigen for

detection of IgG antibodies. In addition it is apparently downregulated during the course of

the disease. Antigen diversity therefore has made the development of immunoblots for use

in Europe far more challenging than in the United States.

Serodiagnosis

Strategies to improve serodiagnosis by using recombinant proteins derived from different

strains have been successful, especially those expressed in vivo like OspC, DbpA, and

VlsE. OspCs and DbpAs have been used for EIAs (Panelius et al., 2003). For commercial

EIAs, recombinant VlsE has been added to increase the sensitivity of conventional EIAs

(Wilske et al., 2007b), and an indirect chemiluminescent immunoassay (CLIA) using

recombinant VlsE based on the EuropeanB. garinii stain PBi is available commercially as

well (see also below).

As in the United States, a two-tiered procedure (Figure 10.1) is recommended for

serodiagnosis in Europe (Wilske et al., 2000). However, the immunoblot interpretation

T A B L E 10.4 Sequence identities among major immmunodominant proteins fromB. burgdorferi sensu stricto, B. afzelii, and B. garinii in Europea

Protein

DNA Sequences

Range (in %)

Amino Acid Sequences

Range (in %)

DbpAb 51–63 40–44

VlsE 65–72 51–56

OspCb 61–77 54–68

OspAb 85–86 78–81

BmpA (p39)b 91–93 89–90

p58b 90–97 90–97

FlaB (flagellin) 94–95 96–97

FlaB fragment (aa 129-251) 92–93 92–96

p83/100b 81–87 87–89

aStrains analyzed were B. burgdorferi B31, B. afzelii PKo, and B. garinii PBi.bSequence identities were calculated without the leader sequence of the lipoproteins.


criteria recommended by the CDC for use in the United States (ASTPHLD, 1995;

CDC, 1995) are not applicable to European patients (Hauser et al., 1997; Wilske

et al., 2007b). Detailed advice on serologic testing for Lyme borreliosis in Europe and

guidelines for the interpretation of standardized immunoblots for the threemain pathogenic

species of borreliae can be found in (Wilske et al., 2000 and 2007b). Reference Wilske

et al., 2000 is available online in English at http://nrz-borrelien.lmu.de/miq-lyme/index.

html. Two presentations of Lyme borreliosis that are unique to European patients are

Borrelia lymphocytoma, an uncommon skin manifestation that occurs early in infection,

and ACA, a chronic late skin condition. The antibody detection rates in patients with

lymphocytoma are 70% to 100% (Pohl-Koppe et al., 1998) andwithACAare 100% (Hansen

and Asbrink, 1989).

Recombinant antigen immunoblots that have been used for years in Europe recentlywere

improved significantly in sensitivity compared with the whole-cell lysate immunoblot by

using a line blot technique and a broad panel of antigens (Wilske et al., 1999; Schulte-

Spechtel et al., 2003; Goettner et al., 2005). For serodiagnosis of acute neuroborreliosis, a

line blot showed a significant increase in sensitivity compared to a whole-cell lysate blot

(Goettner et al., 2005). For this line blot, 7 different borrelial proteins were used. For 5 of

the 7 proteins, 1 to 3 additional homologous proteins from different species were evaluated.

Representative examples are shown in Figure 10.3. Combination of homologues derived

Figure 10.3 Examples of recombinant line IgM and IgG immunoblots. Serum samples are from

three patients with early neuroborreliosis. Borrelia strains belong to the following species: B31 and

PKa2 toB.burgdorferi s.s., PKo toB. afzelii,PBr toB.gariniiOspA-type3, PBi toB.gariniiOspA-type 4,

and 20047 to B. garinii of unknown OspA-type.

HETEROGENEITY OF B. burgdorferi SENSU LATO IN EUROPE 201

from different strains considerably increases the sensitivity of antibody detection, espe-

cially in the case of highly heterogeneous proteins likeDbpA (Table 10.5). This is especially

important in neuroborreliosis where causative strains are very heterogeneous (Panelius

et al., 2003; Schulte-Spechtel et al., 2003; Goettner et al., 2005).

OspC has long been regarded to be the major protein recognized by IgM antibodies. The

line blot study revealed that IgM antibodies reacted comparably well with VlsE derived

from strain PBi. Thiswas not seenwithVlsE fromother strains, suggesting that the source of

the antigen is important in Europe. IgG antibodies reacted preferentially with VlsE of strain

PBi in patients with EM or early neuroborreliosis (Table 10.5). In patients with late

manifestations of Lyme borreliosis (ACA or Lyme arthritis), IgG antibodies reacted well

with VlsE from either B. garinii or B. burgdorferi sensu stricto, but not with VlsE from

B. afzelii, a phenomenon that is difficult to interpret.

NEWER ASSAYS AND FUTURE DIRECTIONS

Clinicians and their patients could all benefit from simpler, more objective, and less costly

alternatives to two-tiered serology. There are many indications that this goal will be

achieved in the not so distant future. The alternatives under consideration are diverse. They

include ”stand-alone” single-step EIAs, bead-based assays using peptides or purified

recombinant antigens and luminometer technology to detect antibodies, and stand-alone

immunoblots striped with defined antigens (either isolated from Borrelia or prepared as

recombinants) and objectively scored by densitometry. New immunoblot interpretation

criteria have been proposed for blots that include VlsE. New methods of data analysis are

being developed to extract additional information thatmay be clinically useful from existing

test results. Novel target antigens or combinations of antigensmay be revealed by proteomic

approaches that have the power to examine the diagnostic utility of each of the proteins

encoded by B. burgdorferi.

At this time the most highly developed and evaluated alternatives to two-tiered serology

are EIAs based on the C6 peptide of VlsE (Liang et al., 1999). The peer-reviewed scientific

literature describes the performance of many C6 peptides assays developed by individual

investigators in their own laboratories, although an increasing number of reports evaluate

the commercial C6 test produced by Immunetics (Marangoni et al., 2005; Cinco and

Murgia, 2006; Smismans et al., 2006; Tjernberg et al., 2007). Accordingly there are

differences between C6 test designs and in the performance characteristics that have been

reported. Depending in part on whether the developer envisioned a C6 EIA as a first-tier

assay or as a stand-alone test that would not require supplementary immunoblotting, the

specificity may be very high (99% or greater) or much lower (92%) (Liang et al., 1999;

Bacon et al., 2003; Peltomaa et al., 2004; Smismans et al., 2006). Despite variability in tests,

such as the cutoff value for a positive result, a consistent picture has emerged that C6 is a

very important diagnostic antigen.

C6 EIAs have been reported to be significantly more sensitive in early acute Lyme

disease in North American patients than conventional two-tiered serology and equivalent to

it in sensitivity in later disease (Bacon et al., 2003, Tables 1 and 2; G. Wormser, personal

communication, 2008). In the United Kingdom, unpublished work suggests that two-tiered

IgM testing may be more sensitive than the commercial C6 assay in early disease (S.

O’Connell, personal communication, 2008). Infections acquired in North America may


TABLE10.5

Antibodyreactivities

with

recombinant

borrelialproteins

inthelineimmunoblot

a.IgG

reactivitieswithDbpA

andVlsE(percent)

DbpA

VlsE

PBi

PBr

PKo

B31

Atleast

PBi

PKo

PKa2

Atleast

Diagnosis

Number

B.garinii

B.garinii

B.afzelii

B.burgdorferi

1DbpA

B.garinii

B.afzelii

B.burgdorferi

1VlsE

EM

a15

013.3

26.7

6.7

33.3

80.0

60.0

40.0

80.0

Early

neuroborreliosis

50

38.0

40.0

34.0

12.0

78.0

88.0

82.0

82.0

92.0

Acrodermatitis

10

10.0

0.0

80.0

10.0

80.0

100.0

0100.0

100.0

Arthritis

10

70.0

10.0

90.0

20.0

100.0

100.0

20.0

90.0

100.0

Controls

110

1.8

1.8

0.0

0.0

3.6

0.9

2.7

0.0

3.6

b.IgM

reactivitieswithOspCandVlsE(percent)

OspC

VlsE

20047

PBi

PKo

B31

Atleast

PBi

PKo

PKa2

Atleast

Diagnosis

Number

B.garinii

B.garinii

B.afzelii

B.burgdorferi

1OspC

B.garinii

B.afzelii

B.burgdorferi

1VlsE

EM

a15

60.0

13.3

73.3

80.0

80.0

53.3

26.7

20.0

53.3

Early

neuroborreliosis

50

20.0

6.0

38.0

52.0

54.0

52.0

14.0

12.0

56.0

Controls

110

0.9

00.9

1.8

1.8

5.4

00

5.4

aDisease

duration>

2weeks.

bB.g.,B.garinii;B.a.,B.afzelii;B.b.,B.burgdorferi.

203

stimulate a more brisk early antibody response than European infections. A C6 assay has

been recommended as the most valuable testing option when early Lyme disease is

suspected in a patient with a “flu-like” illness without EM, an uncommon but difficult

to diagnose situation (Steere et al., 2004). Whether the commercial C6 EIA is approved by

the FDA and adopted as an alternative to two-tiered testing in the United States likely will

depend on whether its specificity is demonstrated to be high enough, especially in late

disease (arthritis, carditis, or neuroborreliosis).

Some investigators are concerned about whether it is wise to rely on a single test

antigen, especially in Europe where multiple species of Borrelia infect patients. Direct

comparisons between the commercial C6 EIA and the leading multi-antigen assays, using

suitably large and diverse serum panels, will be necessary to resolve this matter. In Europe

the single-peptide C6 EIA should be compared with two-tiered serology that incorporates

VlsE in the first-tier EIA and with immunoblots with VlsE and multiple antigens from

different strains.

All three of the major pathogenic species of Lyme disease Borrelia induce antibodies

that recognize the C6 peptide (Liang et al., 2000), despite some sequence differences

between their respective VlsE IR6 regions. This sequence variation opens the question of

whether C6-type assays can be improved by including IR6 variants, particularly for use in

Europe. Some differences in sensitivity have been observed between EIAs constructed with

IR6 variant antigens (Gomes-Solecki et al., 2007; Sillanpaa et al., 2007), but they have not

been described as statistically significant. Indeed the specifics of test design were cited as a

more decisive factor than antigen sequence variation in test results (Sillanpaa et al., 2007).

The titer of antibodies that recognize theC6 antigen declinesmore rapidly after antibiotic

therapy than does the titer of antibodies detected by lysates of whole Borrelia (Philipp

et al., 2001). In a study of antibody responses in patientswith early localized or disseminated

disease, a decrease in titer of four-fold or greater at six months after treatment or a negative

C6 test result was highly associated with a successful outcome of therapy (Philipp

et al., 2001, 2005). Similar observations were subsequently made using the full-length

VlsEmolecule as antigen (Marangoni et al., 2006). The distinctive time course of anti-C6 or

anti-VlsE antibodies after antibiotic therapy for early Lyme disease could assist in the

evaluation of patientswho presentmuch later with a new clinical sign compatiblewith Lyme

disease, for example, facial palsy or meningitis. If the illness is unrelated to infection by

B. burgdorferi, the C6 or VlsE assaymay be negative and thewhole-cell EIA positive due to

the persistence of antibodies elicited by prior Lyme disease that was successfully treated. C6

titers do not wane as rapidly in patients treated for later manifestations of Lyme disease

(Peltomaa et al., 2003), and they may not be useful for evaluating patients with post-

treatment Lyme disease syndrome (Fleming et al., 2004).

Full-lengthVlsE, expressed as a recombinant protein, is the other leading antigen that has

been evaluated as an alternative to two-tiered serology in US patients with encouraging

results. After the initial description of VlsE as a serodiagnostic antigen (Lawrenz

et al., 1999), whole VlsE was identified as the most sensitive and specific of 11 candidate

recombinant antigens for evaluation of early Lyme disease by EIA (Magnarelli et al., 2002).

Indirect chemiluminescent immunoassays (CLIAs) are commercially available based on

recombinant VlsE. In Europe, the Liaison assay produced by DiaSorin contains not only a

VlsE cloned from B. garinii but also recombinant OspC from B. afzelii, bound to magnetic

particles for the analysis of IgG and IgM, respectively (Riesbeck andHammas, 2007). In the

United States, the Diasorin assay contains VlsEs from both B. burgdorferi sensu stricto and

B. garinii but does not include OspC. It has been evaluated (Ledue et al., 2008) and judged


by the FDA to be substantially equivalent to the predicate Immunetics, Inc. C6 assay and, as

noted earlier, has been approved for use as a first-tier assay.

Recent work indicates that C6 andwhole VlsE antigens detect different activities in the

antibody responses to Borrelia infection (Embers et al., 2007). IgG antibodies to C6

peptide were commonly seen in serum samples that lacked detectable IgG reactive with

recombinant VlsE, whereas the converse was rarely observed. In contrast, IgM antibodies

were more frequently reactive with VlsE than with C6 antigen. The frequency of IgG

reactivity alone to the C6 peptide exceeded the combined frequency of either IgM or IgG

reactivity with VlsE in this study of a small number (n¼ 39) of well-characterized

samples. Diagnostic tests based on VlsE may primarily detect antibodies to conforma-

tional epitopes and/or variable regions of the molecule, since the invariant region

reproduced in the C6 assay appears to be largely buried within the protein structure,

and therefore inaccessible to antibody.

APPROPRIATE USE OF SEROLOGIC TESTS

Principles for the use and interpretation of diagnostic data in clinical medicine are well

established and described in numerous texts (e.g., Sackett et al., 1991). The value of

serologic test results depends on their diagnostic predictive values, both positive and

negative. Although much of this chapter has focused on the diagnostic sensitivities and

specificities of various types of serologic assays, it is their predictive values that are most

relevant clinically. In this context the positive predictive value is the probability that a

patient who has a positive test result truly has Lyme borreliosis (expressed as the number of

patients who have Lyme disease out of every 100 patients who have positive test results).

Negative predictive value is the probability that a patient who has a negative test result does

not have Lyme borreliosis (the number of patients who do not have Lyme disease out of

every 100 patients who have negative test results). An assay with high diagnostic sensitivity

improves negative predictive value; one with high diagnostic specificity improves positive

predictive value.

Serologic testing is recommended only for patients who have appropriate pretest

probabilities of Lyme disease in order for the results to have useful predictive values. A

position paper published by the American College of Physicians (ACP) concluded that

laboratory testing should only be done in patients who have a pretest probability of Lyme

disease between 0.20 and 0.80 (Tugwell et al., 1997). The pretest probability is estimated

from the findings of a careful history and physical examination, coupled with familiarity

with the incidence of Lyme disease in the area of residence or travel. In the United States a

practical way of assessing risk by area is to consult the CDC’s national Lyme disease

incidencemap and statistical tables (www.cdc.gov/ncidod/dvbid/lyme/ld_statistics.htm),

which are updated regularly. A map of the average incidence of Lyme by county for the

years 2002 to 2006 is shown in Figure 10.4. The risk of Lyme disease varies greatly by

geographic area. As illustrated, the risk is greatest in the northeast and upper midwest and

is vanishingly small in some areas of the country. Physicians should consult their county

or state health departments for further guidance on the local incidence of Lyme disease,

which can be highly focal.

The ACP panel members pointed out that patients who have only nonspecific signs and

symptoms of illness such as headache, fatigue, muscle or joint pains, even when they

APPROPRIATE USE OF SEROLOGIC TESTS 205

reside in a geographic area endemic for Lyme disease, have a pretest probability of Lyme

disease of less than 0.20, usually much less. Patients with nonspecific findings and no risk

of exposure to infected ticks will have an extremely low pretest probability and certainly

should not be tested. This advice may be contrary to what some health care providers

received during their medical training. At one time it was commonly said that Lyme

disease is one of a group of conditions that can present as almost any syndrome, which we

nowknow is not correct. Routine serologic testing of all patients for whom the diagnosis is

unclear at presentation will result in more false-positive results than true positives. A

negative test result, if testing is performed despite current guidelines, effectively rules out

Lyme disease.

When the pretest probability of Lyme disease is greater than 0.80, laboratory evaluation

adds little useful information. An example of this situation is a finding of EM in a patient in

an endemic area (Tugwell et al., 1997). Tests for antibodies are not sufficiently sensitive for a

negative test result to rule out B. burgdorferi infection.

Serologic testing is of significant value when a patient has objective signs (other than

EM) compatible with Lyme borreliosis (Table 10.2). IgM test results should be used only

within the first month after the onset of initial clinical signs (generally acute neuroborre-

liosis). Positive IgM serology should not be used to support the diagnosis of late

manifestations of B. burgdorferi infection. The effect of serologic results on post-test

Figure 10.4 Average incidence of Lyme disease by county for 2002 to 2006. Cases met the US

national case definition for surveillance and were recorded by state of residence (not state of tick

exposure). Population figures were from mid-year 2004 US Census Bureau estimates. Erythema

migrans–like rashes sometimesoccur after thebites of lonestar ticks (Amblyommaamericanum) and

can be confused with early Lyme disease. Lonestar ticks, which do not transmit Lyme disease

bacteria, are commonhuman-biting ticks in the southernand southeasternUnitedStatesandcanbe

found less commonly in some areas that are endemic for Lyme disease. Source: Centers for Disease

Control and Prevention.


probabilities of Lyme disease in various clinical situations has been calculated and

illustrated (Tugwell et al., 1997). This reference can be highly instructive for the nonspe-

cialist. The clinical signs of neuroborreliosis, Lyme carditis, and Lyme arthritis are

described elsewhere in this volume and are essential to know to estimate pretest proba-

bilities of Lyme disease.

Specific antibodies to B. burgdorferi may be detectable for long periods of time after

successful treatment of Lyme borreliosis (Kalish et al., 2001). Their presence in serum

samples of patients presenting with other conditions, may result in a diagnostic

dilemma. The antibody responses to the C6 synthetic peptide or to recombinant VlsE

decline more rapidly than to whole-cell antigens in patients successfully treated for

early Lyme disease. Assessment of antibodies to C6 or VlsE may be especially helpful

in this situation.

When use of serology is indicated, it is important to select validated laboratory tests.

Health care providers are encouraged to select tests that have been established to be

clinically useful by publication(s) in the peer-reviewed scientific literature, and/or by

approval by regulatory agencies such as the FDA in the United States or by CEmarking, the

minimum standard for commercial diagnostic test kits in Europe (CDC, 2005). In addition

test performance and interpretation should adhere to the recommended guidelines discussed

earlier in this chapter.

ACKNOWLEDGMENT

The authors thank Mr. Mark Pilgard and Dr. Susan O’Connell for critically reading the

manuscript and Ms. Kiersten Kugeler for preparing Figure 10.4.

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